Chemical compounds produced for the use as drug substances, agrochemicals, diagnostics, food additives, flavours, etc. are underlying stringent quality demands in terms of purity or presence of chemical and stereochemical impurities. As typical purity level, a compound with <0.1% impurity is said to be pure in pharmaceutical chemistry. For safety reasons the reporting threshold of impurities in drug substances has therefore been set to 0.05%, which means that analytical procedures adopted for the quality control need to be able to detect and quantify impurities down to this percentage or quantitation limit. Such low quantitation limits are difficult to reach and require assays with high selectivity thereby ensuring their accuracy. Chromatographic methods are often the preferred methodology to do so, in particular in conjunction with highly sensitive and selective mass spectrometric detection.
The impurities which are usually process- and drug-related organic compounds originate from manufacture or storage and may stem from starting materials, by-products of reactions, degradation products, reagents, ligands, catalysts, etc. After the synthesis the products are usually purified by common chemical means such as extraction, crystallization, distillation. However, to end up with above specified high pharmaceutical quality (purity) a final chromatographic purification step is nowadays often employed. In the majority of cases this is chromatography with chemically modified surfaces. The materials which are subject of the present invention comply with both analytical and preparative application and may be employed for basic, neutral, acidic and amphoteric compounds.
One purification problem not yet satisfactorily solved arises in particular for synthetic peptides which are of exceptionally broad use as drugs, drug transporters, diagnostics, radio-pharmaceuticals, synthetic vaccines, bioactive research compounds, building blocks, structural probes, analytical standards, and so forth. In general, the synthesis of peptides, be it by solid-phase synthesis or solution-phase chemistry strategies, provides not only the target peptide, but also impurities which may result from multiple coupling due to deprotection, failed coupling (deleted sequences), hydrolysis of protected side chains (e.g. t-butyl ester of Glu), imide formation, deamidation (e.g. of Gln, Asn side chains), racemization (yielding epimers or diastereomers), oxidation, S-S exchange, β-elimination, and so forth. After the initial standard clean-up procedures these impurities are still present. Hence, a final chromatographic purification step is demanded to end up with the required purity. Nowadays, this final chromatographic purification is normally performed by gradient elution reversed-phase liquid chromatography (RP-HPLC) with acetonitrile as organic modifier (containing 0.1% trifluoroacetic acid) using usually octadecyl-modified silica (ODS) as stationary phase. Although this method offers in principle good selectivity and high efficiency, unfortunately it often fails in particular for very hydrophilic or also very hydrophobic peptides as well as structurally closely related peptides that are often present as impurities. Such side products appear in the RP-HPLC chromatograms as accompanying minor peaks in close vicinity to the front or tailing end of the main component peak, being insufficiently resolved. If it comes to preparative separations where overloading is mandatory to achieve sufficient productivity, they tend to co-elute with the main component which is in particular critical and disturbing when the peptide has to be produced in drug quality (less than 0.1% impurity).
Similar problems and considerations are valid for synthetic oligopeptides of pharmaceutical interest that are also prepared by solid-phase synthesis.
Another major application field of chromatography has become the downstream processing of biopolymers in particular proteins for their purification. Proteins that are chromatographically purified include enzymes, hormones, receptors, transporters, plasma proteins, mono- and polyclonal antibodies, membrane proteins, recombinant proteins, and so forth. The downstream processing, e.g. from a fermentation broth, consists of several steps wherein chromatography is an accepted option even in early purification steps. For example, in the capture step and/or the cell separation expanded bed chromatography, which is a relative new method for the purification of cell broth, may be employed. The separation medium is loosely packed in the column and becomes fluidised during the run. Thus cells can flow through the column while proteins may bind to the separation material. The ionic strength of the medium has to be high enough to prevent cells from binding. Alternatively also inexpensive ion exchange media can be utilized for batch adsorption for this first purification step. The typical purification step is normally done by chromatography using modes like ion exchange, hydrophobic interaction chromatography, affinity chromatography using affinity ligands like complexed metal ions, protein A, protein G, heparin, or dyes. Instead of particulate sorbent beds the use of derivatized membranes is known as interesting alternative (M. Kastner, Ed., Protein Liquid Chromatography, Journal of Chromatography Library, Volume 61, Elsevier, Amsterdam, 2000). Besides, reversed-phase chromatography, gel chromatography, and ion-exchange are frequently employed for final purification (polishing) of protein products or for the virus and endotoxin removal. Often biocompatible conditions are required or at least preferred. This is a considerable drawback of standard RP materials and methods, which require elution conditions that may lead to denaturing. In addition, a low sample loading capacity is typical for RP chromatography like for a number of other protein chromatography modes such as hydrophobic interaction chromatography (HIC) or affinity chromatography like protein A/G as chromatographic ligand. If bioactivity needs to be maintained methods like ion-exchange or affinity chromatography need to be adopted. While the latter has a low capacity, the high sample loading capacity is one of the strengths of ion-exchange. These and other advantages and disadvantages are discussed in detail in textbooks on protein separation (e.g. in M. Kastner (Ed.), Protein Liquid Chromatography, Journal of Chromatography Library, Volume 61, Elsevier, Amsterdam, 2000).
We herein propose a concept for separation media based on various support materials modified with ligands containing both anion exchange sites and binding sites based on non-ionic interactions. The distinct binding domains are integrated in the ligand structure in a sequential combination of the respective structural elements, thus leading to a multi-modal type separation material. The anion exchange sites of the separation materials according to the present invention contain endocyclic nitrogen in cyclic systems with two or more rings as secondary, tertiary or quaternary amine groups. Examples of such ring systems are the quinuclidine or tropane system. This leads to surprising effects such as greatly enhanced selectivities and vastly improved loading capacities compared to materials that utilize the individual chemical interactive groups as surface modifications of carriers. The new separation materials are to be used for the separation and purification of compounds like drugs, drug intermediates, toxins, metabolites of drugs and toxins, fine chemicals, pharmaceuticals, synthetic peptides, biological compounds such as peptides, proteins, RNA, DNA, and carbohydrates, and many others.
As traditionally pointed out the most frequently utilized separation and purification method for drugs, pharmaceuticals and peptides is reversed-phase high-performance liquid chromatography (RP-HPLC). The separation is carried out on materials that possess hydrophobic surface areas or surface layers as formed by long alkyl chains (C8 or C18 phases) or hydrophobic polymers (polymeric RP stationary phases) (for typical examples see H. Schlüter in: M. Kastner (Ed.), Protein Liquid Chromatography, Journal of Chromatography Library, Volume 61, Elsevier, Amsterdam, 2000, p. 157, Table 3.1, or V. R. Meyer, Practice of high-performance liquid chromatography, Salle+Sauerländer, 1990, appendices, summary of commercial columns). The selectivity is based on lipophilicity differences of the solutes to be separated which translates into differential adsorption of the solutes. Some new RP phases have a hydrophilic end-capping or incorporate a hydrophilic group in the allyl strand such as an amide, carbamate or sulfonamide which are then called polar embedded RP phases. These RP phases allow their operation also with purely aqueous eluents and in some instances the polar groups provide an additional retention contribution e.g. for hydrophilic solutes. Such RP type stationary phases are available in a huge variety of variants from a large number of suppliers. They differ from the materials of the present invention by the lack of the anion-exchange site. Likewise, moderately hydrophobic separation materials carrying typically phenyl or C1 to C8 alkyl ligands as interactive moieties which are used for hydrophobic interaction chromatography (HIC) (for examples see L. R. Jacob in: M. Kastner (Ed.), Protein Liquid Chromatography, Journal of Chromatography Library, Volume 61, Elsevier, Amsterdam, 2000, p. 240, Table 4.1) do also not possess an ionic interaction site for (an)ion-exchange. The same applies also to many stationary phases for hydrophilic interaction chromatography (HILIC) that miss this positively charged interactive moiety (anion-exchange site). On the contrary, classical anion-exchange materials such as summarized by P. H. Roos in: M. Kastner (Ed.), Protein Liquid Chromatography, Journal of Chromatography Library, Volume 61, Elsevier, Amsterdam, 2000, p. 18, Table 1.4, miss the hydrophobic long alkyl chain ligand and thus the hydrophobic interaction contribution. On the other hand, conventional anion-exchangers lack also the additional hydrophilic interaction sites which are typical for HILIC materials. Moreover, it is pointed out that the classical anion-exchangers as well as HILIC suitable anion-exchangers are based on non-cyclic amines or in other words are synthesized from non-cyclic, aliphatic amines as building blocks and hence can be clearly distinguished by this criterium from the present invention.
Recently, mixed-modal chromatography which is based on at least two modes of interaction, in most cases ion-exchange and hydrophobic interaction, have become more popular, because it seems that often the achieved resolution outperforms that of corresponding separate individual single-mode chromatographic separations. Such mixed-modal chromatography can be carried out in a number of different variants, which have been reviewed by L. W. McLaughlin (1989) in Chem. Rev. 89, pages 309-319:
Category 1: On-line coupling of different columns packed with individual conventional single mode separation materials that are more or less orthogonal to each other: For example, a reversed-phase column can be on-line coupled to an ion-exchange column. Mixed-modal anion-cation exchange/hydrophilic interaction chromatography was utilized for example by Strege et al. (Anal. Chem., 2000, 72, 4629-4633). The method compares the selectivities obtained by a sequential on-line combination of distinct columns (packed with the individual sorbents viz. anion-exchanger, cation-exchanger, and HILIC material) with the selectivities afforded by the individual columns alone.
Category 2: Mixed-modal chromatography with mixed-bed columns: The blending of distinct separation materials such as ion-exchanger and reversed-phase particle in a single HPLC column leads to mixed-bed columns that may give complementary selectivity compared to columns packed with individual conventional single mode chromatographic particles. For example, blending of two types of different materials such as RP particles and anion-exchanger (e.g. strong anion-exchanger particles) in a single column has been suggested as alternative to combine different retention mechanisms, and such columns are commercially available e.g. with trade name Duet® from Hypersil.
Category 3: The distinct interactive functionalities such as ion-exchanger group and hydrophobic moiety are located on different components of the separation material, i.e. one at the dedicated chromatographic ligand and the other at the support, and thus are spatially separated. Often, the interaction site at the support that is more an undesired residual interactive group rather than a dedicated or customized functional group is not well accessible by the separands thus missing the corresponding selectivity. Such materials are usually obtained, if a support having a specific functionality or physicochemical character such as a hydrophobic nature, is derivatized with another functionality like an ionic group. In such cases, the interactions of the solute with the support are regarded as secondary interactions which are usually assessed to be detrimental and therefore avoided or at least minimized (e.g. by choice of appropriate mobile phase conditions, end-capping, coating or shielding procedures). (Alkyl)amino-modified poly(styrene-co-divinylbenzene) materials are to be classified into this category (C. G. Huber et al., LC-GC, 14, 1996, 114).
Category 4: In another variation, the two complementary interactive functionalities may be present on two distinct spatially separated chromatographic ligands, but randomly immobilized on same support particle leading to a uncontrolled spatial distribution of the two distinct ligands: Such adsorbents on silica basis could be synthesized for instance if a mixture of two different silanes each carrying one of the interactive moieties are used for the immobilization procedure. The combination of two different interaction mechanisms such as anion-exchange and hydrophobic interaction on a single chromatographic particle, but on two distinct interactive ligands (e.g. trimethylammoniumpropyl and C8 or C18 alkyl groups immobilized on silica) has previously been exploited in solid-phase extraction (C. F. Poole, Trends Anal. Chem., 2003, 22, 362-373; M.-C. Hennion, J. Chromatogr. A, 856, 1999, 3-54). Such mixed-modal SPE materials are commercially available from a number of suppliers including Waters (Oasis® MAX) and International Sorbent Technology (Isolute® HAX). Such a type of mixed-modal reversed-phase/anion-exchanger has been specifically developed for capillary electrochromatography (CEC) by copolymerization of two types of monomers one carrying the C18 alkyl group and the other carrying quaternary N-benzyl trimethylammonium groups onto the surface of vinyl-modified silica particles, wherein the ion-exchange site fulfills the function of generation of electroosmotic flow, while the hydrophobic ligand is mainly responsible for selectivity and separation (Scherer et al., J. Chromatogr., A 924,2001, 197-209).
Category 5: The last type of mixed-modal chromatographic material has the two (or more) distinct interaction sites in a single chromatographic ligand.
The present invention provides new types of mixed-modal chromatographic media that belong to the fifth category: distinct interaction sites such as anion-exchange and hydrophobic or hydrophilic moieties in single chromatographic ligand. Documents of prior art relative to the fifth category and commercial products are summarized in the following.
WO 96/09116 discloses mixed modal sorbents which comprise nitrogen containing heteroaromatic bases. Similar mixed modal sorbents are disclosed in WO 00/69872. WO 01/38228 discloses a method for anion-exchange adsorption and anion-exchangers that comprise a base matrix carrying a plurality of mixed modal anion-exchange ligands comprising a positively charged structure and a hydrophobic structure. Among a vast number of alternative ligand structures disclosed in this document some cyclic structures are also mentioned. The same holds true for the disclosure of WO 97/29825.
U.S. Pat. No. 5,147,536 discloses anion exchange materials, whereby the anionic group consists of two positively charged groups at a distance of two atoms from each other. These positively charged groups can be part of an linear structure or of cyclic structures. Among these are derivatives of 1,4-diazabicyclo[2.2.2]octane (DABCO). According to the invention disclosed in this document the spacer, which binds the anionic group to the insoluble support, is supposed not to interact with the sample molecules. Consequently, sorbents comprising derivatives of 1,4-diazabicyclo[2.2.2]octane (DABCO) are not part of the present invention.
WO 02/053252 discloses separation methods using mixed modal adsorbents of various types, some of which are comprise more than one type of ligand bonded to the support, whereby at least one of the ligands can be charged (e.g. positively charged) and is capable for ion-exchange.
Documents disclosing commercialized mixed modal sorbents of the type mentioned above or applications thereof are discussed in the following: An HPLC column (tradename BSC 17 from Cluzeau Info Labo, Saint Foy la Grande, 33220 France) is commercially available from Ehrenstorfer, which is packed with a quaternary anion-exchanger containing a hydrophobic alkyl substituent; the ligand is a 3-(N-dodecyl-N,N-dimethylammonium)-propylsilica (application of this type of material has been described by J.-P. Steghens et al. (2003) J. Chromatogr. B 798, pages 343-349). Similarly, Allsep Technologies offers columns which contain positively charged functional groups derivatized with hydrophobic alkyl groups (tradenames Primesep® B, Primesep® B2). The exact structures are not disclosed, but according to the scheme of the application guide the basic group is a non-cyclic quaternary ammonium ion. D. M. Lubman and coworkers describe the use of a mixed-modal (C18 reversed-phase/anion-exchange) stationary phase that was obtained from Alltech (Deerfield, Ill.) for CEC-mass spectrometry analysis of peptides (Anal. Chem., 71, (1999), pages 1786 ff). According to the authors, the material consists of a spherical silica substrate bonded with a single ligand containing both reversed-phase (C18) and dialkylamine in a fixed 1:1 ratio. Similarly, Hayes and Malik report on the development of silica monoliths with N-octadecyldimethyl(3-trihydroxysilyl-propyl)ammonium chloride ligand in situ incorporated in the sol-gel matrix and their evaluation for CEC (Anal.Chem., 72, (2000), pages 4090 ff.). J. Zhao et al. described a quaternized trimethylaminated polystyrene zirconia as a strong anion-exchange material for HPLC having a mixed-mode retention mechanism with anion-exchange and hydrophobic interaction as well as Lewis acid/base inter-actions (Anal. Chem. 72, (2000), pages 4413-4419). Burton et al. prepared mixed mode sepharose and Perloza bead cellulose matrices carrying hydrophobic and ionic groups obtained by attaching hydrophobic amine ligands to epichlorohydrin (Biotechnology & Bioengineering, (1997), 56, pages 45-55). B.-L. Johansson et al. reported on the synthesis and evaluation of multi-modal anion-exchange separation media with ligands based on aromatic and non-aromatic primary and secondary amines (or both), and in addition hydroxyl groups adjacent to the anion-exchanger site for the capture of proteins at high salt strength from fermentation broth (J. Chromatogr. A, 1016, (2003), pages 21-33). BioSepra® products (of Ciphergen) such as MEP HyperCel® material for hydrophobic charge induction chromatography and MBI HyperCel® material for mixed-mode chromatography combine also more than one separation mechanism on a single ligand. The MEP HyperCel® material is obtained by immobilizing 4-vinylpyridine on mercapto-alkylated chromatographic support by radical addition yielding a 4-[2-(alkylthio)-ethyl]pyridine ligand on the surface of the separation material. The pyridine group is only weakly basic (pKa=4.8). The basic principle is to exploit either hydro-phobic interactions between the ligand (at high pH, pH>pKa i.e. pH>5.8) and the solute which contains both hydrophobic and positively charged groups (designed specifically for biomolecules such as proteins, in particular antibodies) or repulsive electrostatic interactions between the positively charged chromatographic ligand (at low pH, pH< or around pKa i.e. pH between 4-5.8) and the positively ionized solute. The two different types of interactions are typically exploited sequentially viz. adsorption or binding e.g. of antibodies at ‘high’ (around neutral) pH and desorption or elution at low pH (around pKa of pyridine). MBI HyperCel® material for mixed-mode chromatography similarly is based on an aromatic nitrogen-containing group, which in contrast to the MEP HyperCel® material however, carries in addition a strongly acidic sulfonic acid group. The ligand is 2-mercaptobenzimidazole-5-sulfonic acid bonded through the mercapto-group to epoxy-functionalized support by nucleophilic substitution. The resulting material can be classified as a mixed modal cation exchanger. Additional mixed-modal phases such as mixed-modal RP/cation-exchangers (e.g. ABx liquid chromato-graphy column which is a weak cation exchanger: this silica-based mixed-modal ion-exchange matrix was employed for the purification of monoclonal antibodies as described by Ross et al., J. Immunological Anatomy and Biology, (1987), 102, pages 227-231), or immobilized artificial membrane chromatography phases (IAM phases of C.Pidgeon), or mixed-modal zwitterionic HILIC materials (ZILIC, K. Irgum) have been disclosed in literature.
None of the above documents of prior art discloses mixed-modal sorbents with anion-exchange groups based on monobasic cyclic systems with endocyclic nitrogen and with two or more rings as secondary, tertiary or quaternary amine groups like quinuclidine and tropane, as well as their isomers or the more complex structures of Formulae Ic to If.
Quinine and its isomers and derivatives like e.g. quinidine, 10,11-dihydroquinine, or 10,11-dihydroquinidine have been used as chiral effectors (J. Chromat. A. 741 (1996) pages 33-48, and Tetrahedron Asymmetry 14 (2003) pages 2557-2565). In order to achieve good chiral separations sorbents for chiral chromatography are optimized in a way that the chiral interactions are by far the most prominent ones and that other types of non-chiral interactions (e.g. ionic or hydrophobic) are avoided as much as possible. These alkaloids are summarized as cinchona alkaloids. Sorbents comprising such cinchona alkaloids are not part of the present invention.